Abstract

Derivatives of both N-hydroxy-2-acetylaminofluorene (N-OH-AAF) and N-hydroxyphenacetin (N-OH-P) were tested for their ability to cause DNA damage in Reuber (H4-II-E) cells using the alkaline elution technique. Reuber cells are devoid of N-OH-AAF deacylase, N,O-acyltransferase, and sulfotransferase activities. The hydroxamic acids themselves caused very little DNA damage, while N-hydroxy-2-aminofluorine (20 to 100 µm), N-hydroxyphenetidine (20 to 200 µm), and p-nitrosophenetole (10 to 100 µm) all caused dose-dependent damage. The dose-dependent DNA damage caused by N-acetoxy-2-acetylaminofluorene (5 to 25 µm) was completely inhibited by the deacylase inhibitor paraoxon (100 µm). In the presence of both partially purified rabbit liver cytosolic N,O-acyltransferase and guinea pig liver microsomal deacylase, N-OH-AAF was genotoxic. Neither paraoxon nor tRNA had any effect on the DNA damage induced by N-OH-AAF in the presence of N,O-acyltransferase, while paraoxon completely inhibited the damage when N-OH-AAF was incubated in the presence of guinea pig deacylase, and N-OH-P only caused slight DNA damage at higher concentrations of enzyme. In addition, partially purified guinea pig liver deacylase and N-OH-AAF (25 µm) caused 2600 revertants in the Salmonella test system, while only 380 revertants were seen with a 40-fold greater concentration of N-OH-P (1000 µm). The mutagenicity of both N-OH-AAF and N-OH-P was completely inhibited by paraoxon.

Thus, it is clear that metabolites of N-OH-AAF formed outside the cell are capable of passing both the cellular and nuclear membranes to cause genotoxicity. Metabolic activation of N-OH-AAF by either the membrane-bound deacylase or the cytosolic N,O-acyltransferase caused genotoxicity via a deacetylation process. Metabolic activation of N-OH-P by guinea pig deacylase caused low levels of DNA damage, whereas activation by N,O-acyltransferase was not sufficient to cause genotoxicity.

Footnotes

↵1 Pharmacology Research Associate of the National Institute of General Medical Sciences, NIH. To whom requests for reprints should be addressed, at National Cancer Institute, Building 37, Room 3B27, Bethesda, MD 20205.